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letiim bilgisi: akandilci@gyte.edu.tr

E-mail : Alttan alnan dersler belirtilsin. letiim e-mail aracl ile salanacak

Kaynaklar:

* 1.Watson, J.D., et al. Molecular Biology of the Gene. 6/E The Benjamin/Cummings Pub. Co., Menlo Park, California, 2008.

* 2.Alberts, B., et al. Molecular Biology of the Cell. 5/E. ed. Garland Pub., New York,

2008.

Ders deerlendirmesi:

% 50 vizeler ( 2 vize)

% 30 final

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Derse devam

Molekler Genetik I (MBG 222)Yrd. Do. Dr. Ayten Kandilci

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Chapter 2

Nucleic Acids Convey

Genetic Information

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Outline

DNA Can Carry Genetic Specificity

The Double Helix

The genetic information within the DNA is conveyed by

the sequence of its four nucleotide building blocks

The Central Dogma

Establishing the direction of protein synthesis

The era of genomics

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TRANSFORMATION FROM NONVIRULENCE TO

VIRULENCE IS HEREDITARY

Frederick Griffith (British microbiologist)

1928: Non virulent strains of pneumoniae causingbacteria (Streptococcus pneumoniae) become

virulent when mixed with their heat-killed

pathogenic counterparts.

This transformation was hereditary and passed todescendants of newly pathogenic strains.

Without knowing whether or not subsequentgenerations were also virulent, it was also possible

to conclude that the cells became virulent because

they received a factor directly responsible for

virulence, rather than the genetic information

coding for the factor. In that case, however, thevirulence factor would have been diluted upon

division, and would not have been present in

subsequent generations

Frederick Griffith (1879-1941)

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Griffiths experiment raised the

possibility that when pathogenic

cells are killed by heat:

A) Their genetic components

remain undamaged,

B) These components pass

through the wall of recipient

cells and recombine with

recipients genetic apparatus.

(Fred Griffith. The Significance

of Pneumococcal Types. Journal

of Hygiene (1928), 27 : pp 113-

159 )

Frederick Griffith (1879-1941)

TRANSFORMATION FROM NONVIRULENCE TO

VIRULENCE IS HEREDITARY

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Figure 2-1: Transformation of a genetic characteristic of a bacterial cell by

addition of heat-killed cells of a genetically different strain.

TRANSFORMATION FROM NONVIRULENCE TO

VIRULENCE IS HEREDITARY

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Oswald T. Avery, Colin MacLeod andMaclyn McCarty :

1944: Transforming activity of purifiedactive fractions was destroyed by

deoxyribonuclease (DNase; An enzyme that

degrades DNA but has no effect on proteinsor RNA).

Addition of ribonuclease (RNase) or severalproteolytic enzymes had no effect of

transforming activity.

Active genetic component that causestransformation was DNA.

(Oswald T. Avery, Colin M. MacLeod, and

Maclyn McCarty. J Exp Med. 1944

February 1; 79(2): 137158. )

Oswald Theodore Avery (1877-1955)

(Canadian-American microbiologist)

Active Genetic Principle in S. pneumonia is DNA

Colin MacLeod (1909-1972)

(Canadian-American geneticist)

http://upload.wikimedia.org/wikipedia/commons/e/e5/ColinMacCleod.jpg

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Isolation of a chemically pure transforming agent

(figure 2-2)

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Viral Genes Are Also Nucleic Acids

Alfred D.Hershey and Martha Chase (Cold SpringHarbor Laboratory, Long Island, USA);

1952: They labeled the coat and the DNA of bacteriophage T2 with different radioactive

isotopes to see which part of labeled atom of

parental phage entered the host cell and later

appeared in the progeny phage.

Much of the parental nucleic acid but none of theparental protein was detected in the progeny

phage.

Result: Only the DNA component of thebacteriophage T2 carries the genetic information

and the protein coat serves as a protective shell.

(Bacteriophage or phage: Bacterial virus)

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The Double Helix

1938: First X-ray diffraction pattern of

DNA (1938, William Astbury).

1952-1953: High quality X-raydiffraction photographs of DNA

(Rosalind Franklin, Maurice Wilkins )

Suggested that DNA structure is

helical and it is composed of more

than one polynucleotide chain.

The key X-ray photograph involved in

elucidation of DNA structure

(Taken by Rosalind Franklin) (Fig.2-4)

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Chargaffs Rule

1949: Erwin Chargaff(Biochemist);

Examined the relative proportions of adenine, cytosine, guanine, and thymine in

DNA . He Discovered that:

a. the different nucleotides were not all

present at the same concentrations in DNA;

b. their relative levels differed in different

organisms;

c. and that adenosine and thymine, andcytosine and guanine, are present at similar

levels (A=T and G=C).

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Phosphodiester bonds link

together the nucleotides of DNA

1952: Alexander Todd

s group showed

that 3-5 phosphodiester bonds link

together the nucleotides of DNA.

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The Double Helix

1953: Francis H. Crick and James D. Watson

discovered the double-helical structureof DNA (published in Nature, April 25,

1953).

Francis H. Crick

(1916-2004)

James D. Watson

(1928- )

Crick, Watson and Maurice Wilkins awarded the 1962 Nobel Prize for Physiology

or Medicine, "for their discoveries concerning the molecular structure of nucleic

acids and its significance for information transfer in living material."

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1956: Arthur Kornberg haddemonstrated DNA synthesis in cell-free extracts of bacteria. He showed

that a specific polymerizing enzyme

was needed to catalyze the linking of

precursors of DNA.

DNA precursors (nucleotide buildingblocks of DNA):

-dATP (deoxyadenosine-triphosphate)

-dTTP (deoxythymidine-triphosphate)

-dCTP (deoxycytidine-triphosphate)-dGTP (deoxyguanosine-triphosphate)

Finding The Polymerase That Make DNA

Figure 2-7: The nucleotides of DNA

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Enzymatic synthesis of a DNA chain catalyzed by DNA Pol I

The polimerizing enzyme DNA polymerase I (DNA Pol I) links the nucleotides by

3-5phosphodiester bonds. DNA Pol I depends on a DNA template to determine the sequence of the DNA

it is synthesizing.

Figure 2-8

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Proposed models of DNA replication

A) Dispersive (non-conservativemodel): DNA strand is broken in small

pieces and used to prime for

synthesis of new DNA. Than these

pieces are joined together.

B) Semiconservative model: Singlestrand of DNA is conserved during

replication and each parental strand

is distributed into each of the two

daughter strands.

C) Conservative model : Both of the

parental strains remain together and

the two new strands of DNA form an

entirely new DNA molecule.

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DNA strands separate from each

other during replication

1958: Matthew Meselson andFranklin W. Stahl:

They labelled the parental anddaughter DNA with heavy 15N and

light 14N isotopes, respectively.

Separated the heavy (15N- 15N), light(14N- 14N) and heavy+light (15N-14N)

hybrid DNA by centrifugation in

density gradients of heavy salt

cesium cloride.

This experiment showed that doublehelix permanently separate from

each other during replication and

DNA replication is semiconservative.

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DNA replication is a semiconservative process

A) Dispersive (non-conservativemodel): DNA strand is broken in small

pieces and used to prime for

synthesis of new DNA. Than these

pieces are joined together.

B) Semiconservative model: Singlestrand of DNA is conserved during

replication and each parental strand

is distributed into each of the two

daughter strands.

C) Conservative model : Both of the

parental strains remain together and

the two new strands of DNA form an

entirely new DNA molecule.

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Evidence That Genes Control Amino Acid Sequences in Proteins

Wild-type (WT) hemaglobin (Hb)molecules contains alpha and beta

globin chains, which are encoded by

different genes.

Sickle-cell anemia: Individuals have

beta-globinS

allele.

1957: Vernon M. Ingram showed thatS-Hb differs from normal Hb only by

one amino acid (aa).

Because this change in aa sequencewas observed only in patients with

the S-allele of the gene, it was

hypothesized S-allele of the gene

encodes the change in the beta

globin gene.

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DNA Cannot Be the Template That Directly Orders Amino Acids

During Protein Synthesis

In eukaryotic cells;

Protein synthesis occurs at sites where DNA is absent

Protein synthesis in all eukaryotic cells occurs in the

cytoplasm, which separated by nuclear membrane from thechromosomal DNA.

Therefore, at least for the eukaryotic cells, a secondinformation-containing molecule had to exist that obtains its

genetic specifisity from DNA.

This molecule would then move to cytoplasm to function asthe template for protein synthesis.

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The Central Dogma

1953: The working hypothesis was adopted that chromosomal DNA functions

as the template for RNA molecules, which subsequently move to

cytoplasm, where they determine the arrangement of amino acids within

protein.

1956: Francis Crick referred to this pathway for the flow of genetic

information as the central dogma.

Duplication DNA RNA Protein

Transcription Translation

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Discovery of transfer RNA (tRNA)

The discovery of how proteins aresynthesized required the

development of cell-free extracts

capable of making proteins from

amino acids.

These were first effectivelydeveloped beginning in 1953 by Paul

C. Zamecnik and his collaborators.

They discovered that prior to theirincorporation into proteins, amino

acids are first attached to what wenow call transfer RNA (tRNA).

tRNA accounts for about 10% of allcellular RNA.

Figure 2-14: Yeast alanine tRNA structure

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Discovery of Messenger RNA (mRNA)

(Only a few percent of cellular RNA is mRNA)

Bacteria infected with phage T4 provided the

ideal system to find the true template forprotein synthesis.

E.Coli cells infected with phage T4 stopsynthesizing E. Coli RNA; the only RNA

synthesized is transcribed off the T4 DNA.

This T4-RNA does not contribute to ribosomalstructure but it attaches and move across the

ribosomal surface to bring its bases into

positions where they can bind to tRNA-amino

acid precursors for protein synthesis.

T4 RNA orders the amino acids; thus it is thetemplate for protein synthesis.

Since it carries the information from DNA toproteins, it is called messenger RNA (mRNA).

Figure 2-15: Transcription and translation

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Enzymatic Synthesis of RNA upon DNA template is Catalyzed by

RNA Polymerase

1960: Jerard Hurwitz and Samuel B. Weiss

discovered the RNA polymerase.

It synthesize RNA upon DNA templateusing RNA precursors:

ATP, UTP, GTP and CTP. In every enzymatic synthesis, the RNA

AU/GC ratio is similar to the AT/GC

ratio of the template DNA (evidence

that DNA lines up the correct

ribonucleotide precursors).

Synthesis of RNA always begins at 5end and concludes with the 3-end

nucleotide.

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RNA IS SYNTHESIZED IN THE NUCLEUS AND MOVES TO THE

CYTOPLASM

Evidence for the postulated movement of RNA from the DNA-

containing nucleus to the ribosome-containing cytoplasm camefrom the following experiment:

Cells were exposed to radiactive cytidine for 12 minutes with (a pulsechase experiment),

Then allowed to grow for 88 minutes in the presence of excess

amount of unlabeled ribonucleotides (with this experiment, mRNAsynthesized during a short time period was labeled).

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Establishing the Genetic Code

1960s (Charles Yanofsky and Sydney Brenner): Successivegroup of nucleotides along a DNA chain code for successive

amino acids along a given polypeptide.

1961 (Brenner and Crick): Groups of three nucleotides areused to specify individual amino acids.

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Which specific groups of three bases

(codons) determine which specific amino

acids?

1961 (Marshall Nirenberg and Heinrich

Matthaei):

Addition of polynucleotide poly U(UUUUUU) to a cell free system capable

of making proteins leads to the synthesisof polypeptide chains containing only the

phenyl alanine

The nucleotide group UUU must specifyphenyl alanine.

Completion of the code in 1966 showed

that 61 out of 64 permuted groups (43)

corresponds to amino acids (aa), and

most aa are encoded by more than one

nucleotide triplet.

Establishing the Genetic Code

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START AND STOP SIGNALS OF TRANSLATION ARE ALSO

ENCODED WITHIN DNA

Translation both starts and stops at internal positions in mRNA

These start and stop signals that initiate and terminate translation arepresent within DNA (and its mRNA products):

UAA, UAG, UGA (non sense codons or STOP codons)

AUG (methionin, START codon)

In prokaryotes, AUG codons that start new polypeptide chains arepreceded by specific purin-rich blocks of nucleotides that serve to

attach mRNA to ribosomes.

In eukaryotes, the position of AUG relative to the beginning of themRNA is critical determinant, and first AUG is always selected as the

start site of translation.

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